HIV-1 protease, which is required for virion maturation and proliferation, exists naturally as a homodimeric aspartyl protease in which each monomer provides an active site aspartic acid. As expected, site-directed mutation in which the catalytic Asp25 is changed to Asn results in a completely inactive protein complex. In tissue culture experiments, Asp25Asn acts as a dominant negative inhibitor of wild type protease and prevents proteolytic processing and maturation of the HIV virion. A second generation of defective monomers was designed using computer modeling to improve the strength of the dominant negative inhibitor. A resulting variant, Asp25Lys/Gly49Trp/Ile50Trp, has been shown in vitro as well as in vivo to inhibit proteolysis more effectively than Asp25Asn alone. In addition, in vitro studies have demonstrated the triple mutant to form heterodimers with wild type that are more stable than the wild type homodimer, as predicted. The development of HIV-1 protease variants that aggressively form heterodimers with wild type monomers presents a potential new class of macromolecular drugs that may be highly effective against a variety of strains of HIV, including protease-resistant strains. In addition, we are studying a multi-drug resistant variant of HIV PR. This protease, encoded by a viral strain that was transmitted from one host to another despite resistance to numerous drugs, allows us to analyze individual contributions to multiple-drug resistance within a single variant. We plan to study this variant both using traditional molecular biology and kinetic analyses as well as through computational modeling of the amino acid substitutions that contribute to resistance.